Assay for human DNA for gender determination

ABSTRACT

A method for determining gender from a human DNA sample. The loci of Alu element insertion is selected, amplified and evaluated in terms of size of the fragment. The gender assay utilizes AluSTXa for the X chromosome, AluSTYa for the Y chromosome, or both AluSTXa and AluSTYa, to reduce the possibility of error to a negligible quantity. The inserted chromosome yields a large fragment when the homologous region is amplified. The males are distinguished as having two DNA amplicons present, while females have only a single amplicon. The kit adapted for carrying out the method includes a pair of primers to amplify the locus and optionally polymerase chain reaction regents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 10/673,854 filed on 30 Sep. 2003. This relatedapplication is relied on and incorporated herein by references in itsentirety.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention concerns an assay for determination of gender fromhuman DNA samples. More specifically, the invention concerns a processfor determining gender by amplifying an inserted Alu sequence in ahomologous X-Y region of human DNA using appropriate primers, and thendetermining the gender associated with the DNA by determining the lengthof the fragments; the invention also concerns compositions adapted foruse with the process.

2. Description of the Related Art

DNA Typing

DNA (Deoxyribonucleic acid) typing is commonly used to identify theparentage of human children, and to identify the source of blood,saliva, semen, and other tissue found at a crime scene or other sitesrequiring identification of human remains. DNA typing involves theanalysis of alleles of genomic DNA with characteristics of interest,commonly referred to as “markers.” Most typing methods in use today arespecifically designed to detect and analyze differences in the lengthand/or sequence of one or more regions of DNA markers known to appear inat least two different forms in a population. Such length and/orsequence variation is referred to as “polymorphism.” Any region (i.e.“locus”) of DNA in which such a variation occurs is referred to as a“polymorphic locus.”

Genetic markers which are sufficiently polymorphic with respect tolength or sequence have long been sought for use in identityapplications, such as paternity testing and identification of tissuesamples collected for forensic analysis. The discovery and developmentof such markers and methods for analyzing such markers have gone throughseveral phases of development over the last several years.

By the early 1990s, the use of polymerase chain reaction (PCR)technology (disclosed in U.S. Pat. No. 4,683,202 (1987)) was combinedwith the analysis of loci. See K. Kasai et al., Amplification of aVariable Number of Tandem Repeats (VNTR) Locus (pMCT118) by thePolymerase Chain Reaction (PCR) and Its Application to Forensic Science,J. FORENSIC SCI. 35(5):1196-1200 (1990). The amplified products areseparated through agarose or polyacrylamide gels and detected byincorporation of radioactivity during the amplification or bypost-staining with silver or ethidium bromide. However, PCR can only beused to amplify relatively small DNA segments reliably, i.e. onlyreliably amplifying DNA segments under 3,000 bases in length. See M.Ponce et al., PCR amplification of long DNA fragments, NUCLEIC ACIDSRES. 20(3):623 (1992); R. Decorte et al., Rapid Detection ofHypervariable Regions by the Polymerase Chain Reaction Technique, DNAAND CELL BIOL. 9(6):461-469 (1990).

In recent years, the discovery and development of polymorphic shorttandem repeats (STRS) as genetic markers has played an important role inDNA typing. In this approach, amplified alleles at each selected locusmay be differentiated based on length variation. Amplification protocolswith STR loci can be designed to produce small products, generally from60 to 500 base pairs (bp) in length, and alleles from each locus areoften contained within a range of less than 100 bp. This allowssimultaneous electrophoretic analysis of several systems on the same gelor capillary electrophoresis by careful design of PCR primers such thatall potential amplification products from an individual system do notoverlap the range of alleles of other systems.

Gender Assays

Determination of gender from human DNA samples is a common problem inforensics laboratories and in prenatal gender determination. Whileseveral assays based on use of the polymerase chain reaction (PCR) arecurrently available for human sex typing, each of the current approacheshas limitations.

Methods exist that are based on male-specific amplification, such as theamplification of the SRY locus. See A. H. Sinclair, et al., A Gene fromthe Human Sex-Determining Region Encodes a Protein with Homology to aConserved DNA-Binding Motif, NATURE 346:240-244 (1990). These methods,however, lack an internal positive control to discriminate betweenfemale DNA and male DNA which has failed to amplify for technicalreasons.

Restriction fragment length polymorphism (RFLP) assays can be based onsex-specific mutations at the ZFX/ZFY. See R. Reynolds, et al., GenderDetermination of Forensic Samples Using PCR Amplification of ZFX/ZFYGene Sequences, J. FORENSIC SCI. 41:279-286 (1996). RFLP assays,however, require a second enzyme digestion or hybridization stepfollowing the initial PCR amplification.

A recent method proposed by Cali, et al., INT. J. LEGAL MED. 116:133-138(2002), is based on a single adenine insertion within a tandem repeatarray at the DXYS156 locus. But this assay requires access to alleledetection equipment potentially unavailable to forensics labs withlimited resources.

A very widely used approach is based on the Amelogenin locus, whichyields different sized PCR amplicons for the X and Y chromosome versionsof the Amelogenin gene. See K. M. Sullivan, et al., A rapid andquantitative DNA sex test: fluorescence-based PCR analysis of X—Yhomologous gene amelogenin, BIOTECHNIQUES 15:636-638, 640-631 (1993).However, this method misidentifies males as females in some cases (afrequency of 0.018% in Caucasian males, 1.85 % among Indians, and ashigh as 8% in Sri-Lankans) due to a deletion in the AMEL Y region. SeeF. R. Santos, et al., Reliability of DNA-Based Sex Tests, NAT. GENET.18:103 (1998); M. Steinlechner, et al., Rare Failures in the AmelogeninSex Test, INT. J. LEGAL MED. 116:117-120 (2002); K. Thangaraj,et al., Isthe Amelogenin Gene Reliable for Gender Identification in ForensicCasework and Prenatal Diagnosis?, INT. J. LEGAL MED. 116: 121-123(2002). While the frequency of the deletion is relatively low, thecrucial nature of forensic test results in circumstances such as rapeand prenatal gender determination where there is risk for male-specificinherited disorders, makes any source of error a legitimate cause forconcern. This has led several researchers to recommend that Amelogeninnot be relied upon as the sole determinant of gender. See Santos, supra;Steinlechner, supra; Thangaraj, supra; B. Brinkmann, Is the AmelogeninSex Test Valid?, INT. J. LEGAL MED. 116:63 (2002).

Alu Elements

Alu elements are transposable genetic elements which have amplifiedthroughout primate evolution and now comprise roughly 10% of the humangenome. Alu insertions are generally considered to be homoplasy-freewith respect to human population genetics, as the probability of two Aluelements independently inserting in the same genomic location isextremely small. See M. A. Batzer, et al., Alu Repeats and Human GenomicDiversity, NAT. REV. GENET. 3:370-379 (2002).

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a humangender assay that does not have the shortcomings or limitations ofcurrently available assays.

It is another object of the present invention to provide an improvedmethod for determining gender from a human DNA sample.

It is yet another object to provide a method for quantitation of DNA inthe DNA sample.

It is also an object to provide an improved kit for genderdetermination.

In order to achieve the above and other objectives, the preferredembodiment of the present invention is that, for determining gender froma human DNA sample, the method includes the steps of providing a humanDNA sample containing X chromosomal material and potentially containingY chromosomal material, selecting at least one locus from anon-combining X—Y homologous region into which a monomorphic Alu elementhas been inserted during human evolution, amplifying the DNA sample inan amplification reaction, wherein the product of the reaction is amixture of amplified alleles from the amplified locus present in thesample, and determining the gender of the DNA sample by evaluating theamplified alleles in terms of size and number.

The assays of the invention preferably utilize PCR amplification of aDNA sequence of a non-recombining X—Y homologous chromosomal region.Consequently, when this locus is amplified, the result is a largeramplicon associated with the respective X or Y chromosomal region intowhich the monomorphic Alu element has been inserted. Thus, relativeamplicon size provides the reference for gender identification. Thepreferred embodiment of the gender assay utilizes AluSTXa for the Xchromosome and AluSTYa for the Y chromosome. A more preferred embodimentutilizes both AluSTXa and AluSTYa, to reduce the possibility of error toa negligible quantity. Another embodiment of the gender assay mayutilize Y-90 locus.

A kit for determining gender according to the above method comprisespolymerase chain reaction regents which comprise a polymerase andbuffer, and the pair of primers to amplify the locus.

The quantitation of DNA in the mixture sample can be achieved byanalysis for products from the loci, for example, Y-90, AluSTYa and/orAluSTXa. For example, the amount of male DNA in the unknown sample iscomputed by comparing the intensity of the signal from unknown sampleswith the intensity of standard male DNA or from the calibration curve,which can be generated from the results for the standard male DNAsamples.

BRIEF DESCRIPTION OF DRAWINGS

A more complete appreciation of the invention, and many of the attendantadvantages thereof, will be readily apparent as the same becomes betterunderstood by reference to the following detailed description whenconsidered in conjunction with the accompanying drawings in which likereference symbols indicate the same or similar components, wherein:

FIG. 1 is a diagram showing how PCR amplification yields different sizedfragments for an inserted Alu sequence in a homologous X—Y region; and

FIG. 2 shows resolution of fragments on gel after PCR amplification inaccordance with the invention.

DETAILED DESCRIPTION

The inventors determined on using the strategy of seeking an insertionof an Alu element into a non-recombining X—Y homologous region to createa means for differentiating between inserted and non-insertedchromosomes based on PCR amplicon size. While some recently integratedAlu insertions remain polymorphic in the human population, manyultimately reach fixation for the presence of the Alu insertion. Thus,the inventors hypothesized that locating fixed insertions on either theX or Y chromosome could provide a way of identifying the respectivechromosome, as it was ascertained (see FIG. 1) that the insertedchromosome yields a larger fragment when the homologous region isamplified. The amplification is preferably by PCR although otheramplification schemes are now being developed and can be used.

FIG. 1 is a schematic diagram of human sex-typing based upon theanalysis of recently integrated Alu elements. In the diagram, an Aluinsertion has occurred on the Y chromosome within an X—Y homologousregion. Once fixed in the population, the Alu insertion sequence resultsin a larger amplicon on the Y chromosome, allowing for thedifferentiation of the sex chromosomes via PCR amplification. Xchromosome-specific insertions function in the same manner.

By screening X—Y homologous Alu insertions for levels of insertionpolymorphism, the inventors identified that two monomorphic Aluinsertions meet the necessary criteria for a gender determination assayand are preferred. One is fixed on the X chromosome, AluSTXa, and one isfixed on the Y chromosome, AluSTYa. Both of the Alu elements presumablyinserted and reached fixation in the human lineage prior to theradiation of modern humans from Africa. The inventors selected theseloci for use in their gender assay.

The inventors then amplified DNA samples from 778 diverse(African-American, European-American, and Hispanic-American) individualsof defined sex from paternity/identity cases for both the AluSTYa andAluSTXa loci. The assays showed 100% accuracy in gender identification.The DNA samples used in the study consisted of 389 females (278African-American, 102 European-American, and 9 Hispanic-American) and389 males (288 African-American, 90 European-American, and 11Hispanic-American). The statistical likelihood of 100% accuracy ingender identification of this large sample, on a random basis, isinfinitesimal.

Amplification of the loci was conducted via a PCR reaction and fragmentswere resolved on a 2% agarose gel, as shown in FIG. 2. FIG. 2 is anagarose gel chromatograph that shows the results of the mobile elementbased sex typing assay. In FIG. 2, an agarose gel chromatograph from theanalysis of twenty-four individuals using the genetic systems (a)AluSTXa and (b) AluSTYa is shown. Males are distinguished by thepresence of two DNA fragments, while females have a single amplicon. F(female) and M (male) above each sample indicate the known gender.Individual PCR amplifications were performed in 25 μl (microliters)reactions using 25 ng of template DNA, 0.2 μM of each oligonucleotideprimer, 200 mM deoxynucleotide-triphosphates, 1.5 mM MgCl2, 10 mMTris-HCl (pH 8.4) and Taq® DNA polymerase (1 unit). Each sample wassubjected to the same amplification cycle, as follows: initialdenaturation of 150 seconds at 94° C., 32 cycles of one minute ofdenaturation at 94° C., one minute at the specific annealing temperature(58° C. for AluSTYa and 60° C for AluSTXa), one minute of extension at72° C., followed by a final extension at 72° C. for 10 minutes. Foranalysis, 20 μl of the PCR products was fractionated on a 2% agarose gelwhich contained 0.25 μg/ml of ethidium bromide. PCR products werevisualized using ultra violet (UV) fluorescence.

It may be possible to use FMBIO® image analysis system which utilizeslaser scanning to read fluorescence for a high degree of sensitivity andresolution. Because one scanning allows for simultaneous extraction ofdual wave lengths, efficiency in analysis may be improved. It may bealso possible to use Real-time PCR system such as Applied Biosystems7300 Real-time PCR system for fast, quantitative results without gels.

The primers used for the Y insertion, AluSTYa, were (SEQ ID NO: 1)Forward 5′-ACTGCAGTCCGCAGTCCGG-3′ and (SEQ ID NO: 2) Reverse5′-CCACTGACATATTATCAGCCTTATTAC-3′,

yielding an Alu filled site (Y chromosome) fragment of 528 bp and anempty site (X chromosome) fragment of 199 bp. Primers for the Xinsertion, AluSTXa, were (SEQ ID NO: 3) Forward5′-TGAAGAAATTCAGTTCATAGCTTGT-3′ and (SEQ ID NO: 4) Reverse5′-CAGGAGATCCTGAGATTATGTGG-3′,yielding an inserted (X chromosome) fragment of 878bp and an empty site(Y chromosome) fragment of 556 bp.

A primer pair adapted for assaying a Y-chromosome-specific region in asex determination of human cells may be: (SEQ ID NO: 5) Forward5′-AAACATGGCATGGGCCTCT-3′ (SEQ ID NO: 6) Reverse5′-TTTTAAAGCCACAGACCTCTCTCT-3′.

With this primer pair, it is possible to amplify alleles of Y-90 locuson a Y chromosome. With a kit including this primer pair, analysis ofalleles of Y-90 locus on a Y chromosome may be possible.

It will be apparent to those skilled in the art that some variations ofthese primers will also serve effectively, for example, adding ordeleting one or a few bases from the primer and/or shifting the positionof the primer by one or a few bases. Thus, equivalents of the foregoingprimers, and thus primers encompassed by the present invention, includethe primers specifically listed as well as modifications of theseprimers as described hereinabove.

For both of the selected loci, males are distinguished as having two DNAfragments present, while females only have a single fragment (see FIG.2).

Combining these loci together for human gender identification providesan assay with increased accuracy for sex typing, since local deletionsor other types of mutations that eliminate PCR would have to occur in atleast two independent genomic locations to defeat the test. The speedand ease of agarose-based genotyping due to the ˜300 bp differencebetween filled and empty alleles also enhances the utility of the assayin forensic laboratories.

This approach is also amenable to fluorescence-based amplicon detection,and quantitative PCR to resolve male and female contributions tosex-mixed samples. Furthermore, similar approaches based on ascertainingrepetitive element insertions located in homologous sex chromosomeregions will be useful for gender determination in other taxa ofheterogametic sex. The invention is considered to extend generically tothe use of other loci of X-Y homologous regions where the insertedchromosome yields a large fragment when the homologous region isamplified.

The quantitation of male DNA in the mixture sample can be achieved byanalysis for products from the loci, for example, AluSTYa or AluSTXa, orY-90. The amount of male DNA in the unknown sample is computed bycomparing the intensity of the signal from unknown samples with theintensity of standard male DNA or from the calibration curve, which canbe generated from the results for the standard male DNA samples.

Methods for Performing Assays EXAMPLE 1

Stock primers were reconstituted in sterile TLE to a concentration of100 μM. Then 1 ml of working solution of each primer at 2 μM was made bydiluting 20 l of each stock with 980 μl of TLE. This represents a 10×working concentration of each primer. PCR reagents were prepared on ice.5 μl of DNA template (at 5 ng/μl) or controls was pipetted into eachappropriate well. Then a master mix of all remaining PCR reagents wasprepared. (See Table 1). 20 μl of master mix was pipetted into each wellto make a final PCR reaction volume of 25 μl. PCR tubes or a plate wereinserted into a thermal cycler. TABLE 1 volume example used for 20 Finalconcentration of PCR reagents: (μl) samples PCR buffer (10×) 1× 2.5 50Forward primer: 0.2 μM 2.5 50 Reverse primer: 0.2 μM 2.5 50 dNTPs: 200μM each 0.2 4 MgCl2: 1.5 mM 1.5 30 Cresol red based dye (see suppliesand solutions) 2.5 50 Taq ®DNA polymerase: 1 unit (i.e. 5 U/μl) 0.2 4Sterile water: balance to 20 μl reaction volume 162 Final volume (20 μl× 20 samples) = 400

PCR products were resolved by an agarose gel electrophoresis. 5 grams ofagarose was placed in a bottle. 1×TBE (Tris/Boricacid/EDTA(ethylenediaminetetraacetic acid)) was added to a final volumeof 250 ml (2%=2 g/100 ml). Agarose and TBE were swirled briefly. Thebottle was placed in a microwave oven for approximately 3 minutesrunning time. The contents in the bottle were swirled once or twice asneeded. When the agarose was completely in solution, the bottle wasremoved from the microwave oven. The contents in the bottle were cooled.5 micro liters of ethidium bromide were added to 250 ml of agarose(final concentration 0.2 μg/ml). Then the ethidium bromide and agarosewere swirled to be mixed. The agarose was cooled. The gel was slowlypoured into a gel tray. Combs were added immediately after pouring gel.The gel was solidified for about 30 minutes. The gel tray was placedinto an electrophoresis unit. The gel was covered with 1×TBE. About 20micro liters of PCR product were loaded into each well. Electrophoresiswas performed at 3.5 Volts/cm for at least 1 hour. DNA fragments werevisualized under UV (ultraviolet) light.

EXAMPLE 2 Forensic Assay

The DNA from the source of blood, saliva, semen, and other tissues foundat a crime scene was extracted by standard methods. The extracted DNAwas then amplified for two regions AluSTXa and AluSTYa. The reactionmixture for the amplification for the AluSTXa region was comprised of 25ng of template DNA, 0.2μM of each oligonucleotide primer specific forAluSTXa region as here in described, 200 μMdeoxynucleotide-triphosphates, 1.5 mM MgCl₂, 10 mM Tris-HCl (pH 8.4) andTaq®DNA polymerase (1 unit) in 25μl volume and the amplificationreaction was performed as follows: initial denaturation of 150 secondsat 94° C., 32 cycles of one minute of denaturation at 94° C., one minuteat the 58° C., one minute of extension at 72° C., followed by a finalextension at 72° C. for 10 minutes. Similarly, the reaction mixture forthe amplification for the AluSTYa region was comprised of 25 ng oftemplate DNA, 0.2 μM of each oligonucleotide primer specific for AluSTYaregion, 200 μM deoxynucleotide-triphosphates, 1.5 mM MgCl₂, 10 mMTris-HCI (pH 8.4) and Taq®DNA polymerase (1 unit) in 25 μl volume andthe amplification reaction was performed as follows: initialdenaturation of 150 seconds at 94° C., 32 cycles of one minute ofdenaturation at 94° C., one minute at the 60° C. (or 58° C. forAluSTYa), one minute of extension at 72° C., followed by a finalextension at 72° C. for 10 minutes. The amplified products from thesereactions were analyzed by electrophoresis; 20 μl of the PCR productswas fractionated on a 2% agarose gel which contained 0.25 μg/ml ofethidium bromide. PCR products were visualized using ultra violet (UV)fluorescence. Males are distinguished by the presence of two DNAfragments, while females have a single amplicon.

EXAMPLE 3 Fluorescence-Based Assay

As previously indicated, the inventors' assay can be modified to embodyfluorescence-based amplicon detection and quantitative PCR to resolvemale and female contributions to sex-mixed samples.

The quantitation of male DNA in the mixture sample was performed byanalysis for AluSTYa. A series of known male DNA samples (also called asstandard) are amplified with the unknown sample containing the male DNA.The amount of male DNA in the unknown sample is computed by comparingthe intensity of the signal from unknown samples with the intensity ofstandard male DNA. Alternatively, a calibration curve can be generatedfrom the results for the standard male DNA samples. The quantity of themale DNA in the unknown sample was computed from the calibration curve.

Kits

It is considered that the scope of the invention extends to kits used topractice the assays of the invention. Thus, it is contemplated that theinvention would be exploited by marketing kits for gender determinationof unknown biological samples, using the principles and proceduresdescribed hereinabove. A human gender determination kit comprisesreagents for a polymerase chain reaction, the primers for AluSTYa orAluSTXa, and optionally other reagents for polymerase chain reactionand/or detection.

EXAMPLE 4 Kit for Gender Determination

A kit suitable for performing a single gender determination assaycomprises the following materials: a vial containing 500 ml of XYZsuspended in 5% aqueous NaCl solution; a vial containing 50 ml of GenderDetermination Primer Mix (3 mg NaCl; 10 mg KBr; 14 ml CH₃OH; sufficientdistilled water to bring mix up to 50 ml); a vial containing 10 ml ofReference Solution, and so on.

Several alternative methods for gender determination and quantitationusing Alu insertions have been described above. While the invention hasbeen described in connection with specific and preferred embodimentsthereof, it is capable of further modifications without departing fromthe spirit and scope of the invention. This application is intended tocover all variations, uses, or adaptations of the invention, following,in general, the principles of the invention and including suchdepartures from the present disclosure as come within known or customarypractice within the art to which the invention pertains, or as areobvious to persons skilled in the art, at the time the departure ismade. It should be appreciated that the scope of this invention is notlimited to the detailed description of the invention hereinabove, whichis intended merely to be illustrative, but rather comprehends thesubject matter defined by the following claims.

1. A primer pair for a sex determination of a human DNA sample, saidprimer pair comprising: a forward primer and a backward primer, saidprimer capable of amplifying a selected locus of a human DNA in anamplification reaction, said selected locus being in a non-combining X—Yhomologous region, said region containing a monomorphic Alu insertion inone of the X chromosome and the Y chromosome.
 2. The primer pair ofclaim 1, wherein said selected locus comprises STYa locus.
 3. The primerpair of claim 2, wherein each of the primer pair has the sequenceselected from, or constituting a subset of, the group consisting of:(SEQ ID NO: 1) Forward 5′-ACTGCAGTCCGCAGTCCGG-3′; and (SEQ ID NO: 2)Reverse 5′-CCACTGACATATTATCAGCCTTATTAC-3′.


4. The primer pair of claim 1, wherein said selected locus comprisesSTXa locus.
 5. The primer pair of claim 4, wherein each of the primerpair has the sequence selected from, or constituting a subset of, thegroup consisting of: (SEQ ID NO: 3) Forward5′-TGAAGAAATTCAGTTCATAGCTTGT-3′; and (SEQ ID NO: 4) Reverse5′-CAGGAGATCCTGAGATTATGTGG-3′.


6. A primer pair for a sex determination of a human DNA sample, saidprimer pair comprising: a forward primer and a backward primer, saidprimer pair capable of amplifying alleles of Y-90 locus in anamplification reaction.
 7. The primer pair of claim 6, wherein eachprimer of the primer pair has the sequence selected from, orconstituting a subset of, the group consisting of: (SEQ ID NO: 5)Forward 5′-AAACATGGCATGGGCCTCT-3′; and (SEQ ID NO: 6) Reverse5′-TTTTAAAGCCACAGACCTCTCTCT-3′.


8. A kit for determining gender from a sample, comprising: a polymerasechain reaction reagent; and a pair of primers capable of amplifying aselected locus of a human DNA in an amplification reaction, saidselected locus being in a non-combining X-Y homologous region, saidregion containing a monomorphic Alu insertion in one of the X chromosomeand the Y chromosome.
 9. The kit of claim 8, wherein the selected locusis AluSTYa locus on the Y chromosome.
 10. The kit of claim 9, whereineach primer of the primer pair has the sequence selected from, orconstituting a subset of, the group consisting of: (SEQ ID NO: 1)Forward 5′-ACTGCAGTCCGCAGTCCGG-3′; and (SEQ ID NO: 2) Reverse5′-CCACTGACATATTATCAGCCTTATTAC-3′.


11. The kit of claim 8, wherein the selected locus is AluSTXa locus onthe X chromosome.
 12. The kit of claim 11, wherein each primer of theprimer pair has the sequence selected from, or constituting a subset of,the group consisting of: (SEQ ID NO: 3) Forward5′-TGAAGAAATTCAGTTCATAGCTTGT-3′; and (SEQ ID NO: 4) Reverse5′-CAGGAGATCCTGAGATTATGTGG-3′.


13. The kit of claim 8, wherein the selected locus comprises AluSTYalocus and AluSTXa locus.
 14. The kit of claim 13, wherein said pair ofprimers comprises: a first primer pair, each primer of the first primerpair having the sequence selected from, or constituting a subset of, thegroup consisting of: (SEQ ID NO: 3) Forward5′-TGAAGAAATTCAGTTCATAGCTTGT-3′; and (SEQ ID NO: 4) Reverse5′-CAGGAGATCCTGAGATTATGTGG-3′; and

a second primer pair, each primer of the second primer pair having thesequence selected from, or constituting a subset of, the groupconsisting of: (SEQ ID NO: 1) Forward 5′-ACTGCAGTCCGCAGTCCGG-3′; and(SEQ ID NO: 2) Reverse 5′-CCACTGACATATTATCAGCCTTATTAC-3′.


15. A kit for determining gender from a sample, comprising: a polymerasechain reaction reagent; and a pair of primers capable of amplifyingalleles of Y-90 locus in an amplification reaction.
 16. The kit of claim15, wherein each primer of said pair of primers contains the sequenceselected from, or constituting a subset of, the group consisting of:(SEQ ID NO: 5) Forward 5′-AAACATGGCATGGGCCTCT-3′; and (SEQ ID NO: 6)Reverse 5′-TTTTAAAGCCACAGACCTCTCTCT-3′.